By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg 2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.

en_US

dc.relation.ispartof

Protein Science

en_US

dc.relation.isbasedon

10.1110/ps.04638704

en_US

dc.subject.classification

Biophysics

en_US

dc.subject.mesh

Methyltransferases

en_US

dc.subject.mesh

Phosphotransferases

en_US

dc.subject.mesh

Recombinant Proteins

en_US

dc.subject.mesh

Cloning, Molecular

en_US

dc.subject.mesh

Polymerase Chain Reaction

en_US

dc.subject.mesh

Sequence Alignment

en_US

dc.subject.mesh

Environment

en_US

dc.subject.mesh

Amino Acid Sequence

en_US

dc.subject.mesh

Sequence Homology, Amino Acid

en_US

dc.subject.mesh

Open Reading Frames

en_US

dc.subject.mesh

Molecular Sequence Data

en_US

dc.subject.mesh

Amino Acid Sequence

en_US

dc.subject.mesh

Cloning, Molecular

en_US

dc.subject.mesh

Environment

en_US

dc.subject.mesh

Methyltransferases

en_US

dc.subject.mesh

Molecular Sequence Data

en_US

dc.subject.mesh

Open Reading Frames

en_US

dc.subject.mesh

Phosphotransferases

en_US

dc.subject.mesh

Polymerase Chain Reaction

en_US

dc.subject.mesh

Recombinant Proteins

en_US

dc.subject.mesh

Sequence Alignment

en_US

dc.subject.mesh

Sequence Homology, Amino Acid

en_US

dc.title

New enzymes from environmental cassette arrays: Functional attributes of a phosphotransferase and an RNA-methyltransferase

en_US

dc.type

Journal Article

utslib.description.version

Published

en_US

utslib.citation.volume

6

en_US

utslib.citation.volume

13

en_US

utslib.for

0601 Biochemistry and Cell Biology

en_US

utslib.for

0601 Biochemistry And Cell Biology

en_US

utslib.for

0802 Computation Theory And Mathematics

en_US

utslib.for

0899 Other Information And Computing Sciences

en_US

dc.location.activity

ISI:000221630901

en_US

pubs.embargo.period

Not known

en_US

pubs.organisational-group

/University of Technology Sydney

pubs.organisational-group

/University of Technology Sydney/Faculty of Science

pubs.organisational-group

/University of Technology Sydney/Faculty of Science/School of Life Sciences

utslib.copyright.status

closed_access

pubs.issue

6

en_US

pubs.publication-status

Published

en_US

pubs.volume

13

en_US

Abstract:

By targeting gene cassettes by polymerase chain reaction (PCR) directly from environmentally derived DNA, we are able to amplify entire open reading frames (ORFs) independently of prior sequence knowledge. Approximately 10% of the mobile genes recovered by these means can be attributed to known protein families. Here we describe the characterization of two ORFs which show moderate homology to known proteins: (1) an aminoglycoside phosphotransferase displaying 25% sequence identity with APH(7″) from Streptomyces hygroscopicus, and (2) an RNA methyltransferase sharing 25%-28% identity with a group of recently defined bacterial RNA methyltransferases distinct from the SpoU enzyme family. Our novel genes were expressed as recombinant products and assayed for appropriate enzyme activity. The aminoglycoside phosphotransferase displayed ATPase activity, consistent with the presence of characteristic Mg 2+-binding residues. Unlike related APH(4) or APH(7″) enzymes, however, this activity was not enhanced by hygromycin B or kanamycin, suggesting the normal substrate to be a different aminoglycoside. The RNA methyltransferase contains sequence motifs of the RNA methyltransferase superfamily, and our recombinant version showed methyltransferase activity with RNA. Our data confirm that gene cassettes present in the environment encode folded enzymes with novel sequence variation and demonstrable catalytic activity. Our PCR approach (cassette PCR) may be used to identify a diverse range of ORFs from any environmental sample, as well as to directly access the gene pool found in mobile gene cassettes commonly associated with integrons. This gene pool can be accessed from both cultured and uncultured microbial samples as a source of new enzymes and proteins.